Method of using anticoagulant solution in blood separation

ABSTRACT

An anticoagulant solution of sodium citrate for use in blood chemistry-related techniques and apparatus is disclosed. The anticoagulant solution should include an effective concentration of sodium citrate sufficient for preventing the coagulation of a sample of blood employed in the technique or added to the apparatus. The sodium citrate-based anticoagulant solution should have a pH ranging from above 6.0 to about 8.5 and a sodium citrate concentration preferably ranging from about 0.05M to about 0.2M.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention generally relates to blood chemistry laboratorytechniques and apparatus. More particularly, the present inventionrelates to methods for separating mononuclear cells, such as lymphocytesand monocytes, from whole blood specimens, especially assemblies ormethods which maintain blood in an anticoagulated state prior topartitioning the sample into discrete layers using centrifugation.Additionally, the present invention relates to blood separationassemblies which provide a high recovery of mononuclear cells withoutsignificant contamination by red blood cells.

2. Description of the Prior Art

Considerable research has been conducted in recent years to developimproved methods and devices for separating mononuclear cells, such aslymphocytes and monocytes, from whole blood. Effective separation andisolation of these cells is often critical to various clinical assays aswell as to research laboratory protocols. Consequently, a variety ofblood collection/separation tubes have been developed.

For example, one currently available blood separation device includes ablood collection tube containing an aliquot of a Newtonian gel and analiquot of a liquid density medium, such as Ficoll-Paque™. The Newtoniangel acts as a barrier between the liquid density medium and a sample ofanticoagulated whole blood which is placed in the tube above the gelprior to centrifugation. When the tube is subsequently centrifuged, theliquid density medium acts to separate the mononuclear cells from theother blood components. The separation occurs because the specificgravity of the density medium is greater than the specific gravity ofthe mononuclear cells but less than the specific gravity of the otherblood components.

The aforementioned device and method works well where the tube isimmediately centrifuged upon receipt of the whole blood sample butcannot be utilized as an effective separation means where the bloodsample is placed in the tube and must be shipped elsewhere forsubsequent centrifugation, isolation and analysis. The reason for thislimitation is that both the Newtonian gel and the liquid density mediumare flowable liquids and will not retain their respective positions inthe tube due to movements occurring during shipping. As a result, theblood sample will mix with the density medium during shipping, therebydiluting it. Dilution of the medium adversely affects the properseparation of the blood sample upon subsequent centrifugation at thelaboratory. Such dilution is especially problematic in laboratorystudies where contamination of the mononuclear layer by red blood cellsmust be minimized.

Consequently, other blood separation devices have been developed. Forexample, one of these devices is a blood separation tube in which aporous foam-like plug is deployed above a liquid density separationmedium. The plug functions as a "baffle" for preventing a blood sampleintroduced into the tube from mixing with the separation medium prior tocentrifugation. Although the plug is constructed with a porositysuitable for allowing migration of red blood cells and separation mediumtherethrough during centrifugation, the plug design prevents the bloodsample from interfacing with the separation medium prior tocentrifugation, thereby facilitating a cleaner separation of themononuclear cell layer.

Another currently available blood separation device utilizes anon-Newtonian, thixotropic gel. This gel is positioned within a bloodcollection tube in order to form a stable barrier between the liquiddensity medium and the blood sample prior to centrifugation. Forexample, U.S. Pat. No. 4,867,887 to Smith discloses several embodimentsof a separation device utilizing a thixotropic gel. In one suchembodiment, a blood collection tube includes a layer of a Newtonian gelpositioned in the bottom of the collection tube. A thixotropic gel istemporarily positioned immediately above the Newtonian gel in order toprevent the Newtonian gel from mixing with the blood sample prior tocentrifugation. A space is provided above the thixotropic gel suitablefor allowing placement of a blood sample into the tube prior tocentrifugation.

Upon the addition of an anticoagulated whole blood sample and subsequentcentrifugation, the thixotropic gel moves to the bottom of the tube,thereby displacing the Newtonian gel. Since the specific gravity of theNewtonian gel is less than the specific gravity of the thixotropic gel,the Newtonian gel moves to a new position above the thixotropic gelwhere it mixes with the blood sample and acts as a density separationmedium much like the liquid density medium, Ficoll-Paque™. Red bloodcells, which have the highest specific gravity of any of the componentsin whole blood, are pelleted along with the granulocytes to the bottomof the tube immediately below the thixotropic layer. The mononuclearcells form a layer immediately above the Newtonian gel, therebyrendering an effectively isolated layer of cells to be removed forclinical analysis or other laboratory manipulations.

A further embodiment disclosed in U.S. Pat. No. 4,867,887 to Smithinvolves a similar configuration for a blood separation device includinga collection tube containing a density gradient material, a Newtoniangel, a thixotropic gel and an aliquot of ananticoagulant/cell-sustaining solution. Prior to the addition of anyblood sample or centrifugation, the density gradient material and theNewtonian gel are positioned in the bottom portion of the tubeimmediately below the thixotropic gel. An aliquot ofanticoagulant/cell-sustaining solution is placed into the tube above thethixotropic gel which serves as a temporary barrier to isolate thesolution from the other components in the tube. A space is providedwithin the tube above the solution. The space is suitable for allowingthe placement of an anticoagulated blood sample into the tube prior tocentrifugation.

After a sample has been received in the tube and centrifugation isperformed, the red blood cells and granulocytes are pelleted to aposition at the bottom of the tube immediately below the thixotropic gellayer. The density gradient material moves to a new position above thethixotropic gel where it mixes with the blood sample as well as theanticoagulant/cell-sustaining solution in order to perform a separationfunction. The density gradient material includes both a light and aheavy phase which separate into two layers in order to sandwich theNewtonian gel layer therebetween. The mononuclear cells form a layerimmediately above the lighter phase of the density gradient and aresubsequently removed for analysis.

In both of the embodiments disclosed by Smith, the thixotropic gel actsas a barrier to isolate the other components of the separation devicefrom one another prior to centrifugation. In the second embodiment, theNewtonian gel is not used as a barrier nor a separation medium since thethixotropic gel and the density gradient material perform thesefunctions, respectively. The Newtonian gel alternatively performs aquality control function in that it provides a sticky surface to retainany residual red blood cells located near the mononuclear cell layerafter centrifugation, thereby facilitating removal of the mononuclearcells and limiting any contamination with residual red blood cells.

U.S. Pat. No. 3,852,194 discloses a simpler blood separation deviceutilizing a thixotropic gel material having a specific gravity inbetween that of mononuclear cells and other components of whole blood,such as red blood cells and granulocytes. Consequently, the thixotropicgel functions as a separation medium rather than performing a barrierfunction as in the '887 patent to Smith.

Other blood separation devices are designed for blood collection as wellas separation. For example, various direct draw blood collectioncontainers are currently available. Some of these are designed forundergoing subsequent centrifugation, thereby eliminating the need formechanically transferring a blood sample to a different containier priorto inducing separation via centrifugation.

The blood collection and/or separation tubes mentioned above are only afew of many such containers known to those skilled in the art.Regardless of the type of container employed to perform the collectionand/or separation function, preventing contamination of the mononuclearcell layer with red blood cells remains a key concern for diagnosticstudies which demand minimal levels of RBC contamination.

Various anticoagulants have been used in blood collection/separationdevices either alone or in conjunction with a cell-sustaining solutionsin order to preserve the blood sample in an uncoagulated state for aperiod of time prior to centrifugation and analysis. For example, somecommon anticoagulants include sodium heparin, K₃ EDTA and sodiumcitrate. In particular, sodium citrate solutions have been used for manyyears as anticoagulants. For example, current requirements for geneamplification technologies, such as the polymerase chain reaction,recommend the use of sodium citrate for performing an anticoagulationfunction in whole blood. See Holodniy, M.; Kim, S.; Katzenstein, D.;Konrad, M.; Groves, E.; Merigan, T. C.; "Inhibition of HumanImmunodeficiency Virus Gene Amplification by Heparin", J. Clin.Microbiol. 29:676-679 (1991).

It is known that calcium plays a key role in the blood coagulationcascade. Sodium citrate solutions prevent the participation of calciumin blood coagulation. Typically, these sodium citrate solutions areadded to freshly collected whole blood to prevent coagulation.Subsequently, calcium can be added back to the whole blood suspension toinduce subsequent coagulation when desired.

Sodium citrate is a particularly advantageous anticoagulant as itprovides good buffering capabilities over a range of pH. In particular,the buffering capability of sodium citrate is attributable to threecarboxyl groups present on the corresponding acid of the compound. Sincesodium citrate is the corresponding sodium-based salt of citric acid, itis actually the citric acid/sodium citrate combination that actuallyfunctions to perform the buffering chemistry.

As mentioned above, citric acid (hydroxytricarboxylic acid) has 3carboxyl groups and consequently 3 pKa's. The first pKa appears at a pHof about 3.06. The second pKa appears at a pH of about 4.74. The thirdpKa appears at a pH of about 5.4. Accordingly, sodium citrate performsits most effective buffering functions at these pH values and isespecially useful in performing buffering functions when added to invitro cell suspensions. Consequently, sodium citrate has been used as ananticoagulant in a variety of blood separation devices due to itsbuffering capability over a range in pH.

In particular, citrate has been commonly used as an anticoagulant inthree types of solutions. The first type of solution is referred to asbuffered sodium citrate. The second type of solution is typicallyreferred to as CPD solution or citrate-phosphate-dextrose. The thirdtype is denoted as ACD or acid-citrate-dextrose. The citrate ionconcentration in these solutions is typically greater than theconcentration needed to perform an anticoagulation function. Examples ofthese three solutions as well as other currently available citrate-basedanticoagulant solutions are included in the following table.

CITRATE-BASED ANTICOAGULANT FORMULATIONS

    ______________________________________    Buffered Sodium Citrate    Per Liter:    0.109 Molar 0.129 Molar    ______________________________________    24.7 gm     32.0 gm     Sodium.sub.3 Citrate.2H.sub.2 O    4.42 gm      4.2 gm     Citric Acid.H.sub.2 O    pH 6.1    Acid Citrate Dextrose    Per Liter:    ACD-A       ACD-B    ______________________________________     0.2 gm      0.2 gm     Potassium Sorbate    (Antimycotic)    22.0 gm     13.2 gm     Sodium.sub.3 Citrate.2H.sub.2 O    24.5 gm     14.7 gm     Dextrose.H.sub.2 O     8.0 gm      4.8 gm     Citric Acid.H.sub.2 O    pH 5.05     pH 5.1    CPD (CPDA-1 Contains 0.275 gm ADENINE)    Per Liter:     0.2         Potassium Sorbate    26.3 gm      Sodium.sub.3 Citrate.2H.sub.2 O    3.27 gm      Citric Acid.H.sub.2 O    2.22 gm      Monobasic Sodium Phosphate.H.sub.2 O    25.5 gm      Dextrose.H.sub.2 O    pH 5.8    Alsever's Solution    Per Liter:     8.0 gm      Sodium.sub.3 Citrate.2H.sub.2 O    22.6 gm      Dextrose.H.sub.2 O     4.2 gm      Sodium Chloride                 Citric Acid to Adjust pH to 6.1    LeucoPREP ™ Citrate    Per Liter:    0.10 Molar    ______________________________________    29.4 gm      Sodium.sub.2 Citrate.2H.sub.2 O    0.27 gm      Citric Acid.H.sub.2 O    pH 7.0    ______________________________________

Typically, the highest pH at which sodium citrate has been used in thepast as an anticoagulant has been 6.0. This pH level constitutes theupper limit within which sodium citrate has been utilized as aneffective buffer. In most blood separation/collection tube devices usedfor separating mononuclear cells from whole blood, any sodium citratesolution incorporated therein to perform an anticoagulation function hashad a pH ranging from about 5.05 to about 6.0. In the past, this rangein pH has been the most preferential.

Unfortunately, the use of various sodium citrate anticoagulantformulations have not provided satisfactory results when used in many ofthe currently available blood separation/collection containers,especially those which utilize a thixotropic gel as a barrier or aseparation means. In particular, the resulting layer of mononuclearcells appearing after centrifugation is contaminated to an unacceptablelevel with red blood cells when any of the above-mentioned sodiumcitrate-based formulations are used as anticoagulants. Thiscontamination is very problematic with respect to certain laboratorymanipulations requiring a high degree of purity in the mononuclear cellfragment.

For example, the mononuclear layer of cells contain lymphocytes whichplay a major part in the body's immune system. They are harvested andused in research activities directed toward defining the molecularbiology and cellular interactions of immune mechanisms. Additionally,the analysis of lymphocytes is important in infectious diseasedetection, in cancer and auto-immune disease research, and, isfundamental to monoclonal antibody technology.

Due to the significance of lymphocytes, isolation of these cells fromhuman blood is necessary for a variety of diagnostic assays. Included insuch assays are functional assays, paternity testing and tissue typing.Additionally, an assessment of immune competency can be accomplishedthrough analysis of lymphocyte sub-types and ratios. Accurateassessments of immune competency is significant in the diagnosis of AIDSand is prognostic in many other chronic and often terminal infections.Cellular assays are also utilized to monitor immune regulating drugsemployed in cancer therapy. Additionally, accurate determination oflymphocyte surface markers is critical for histocompatibilitydeterminations.

As a consequence of the foregoing, it is necessary that methods andapparatus for providing a highly effective separation of mononuclearcells from the other components in whole blood are available toclinicians as well as research investigators. If the mononuclear cellsare contaminated to an unacceptable level with red blood cells orgranulocytes, many of the foregoing objectives cannot be easilyachieved.

For example, it has been discovered that whole blood samples added toblood separation devices utilizing thixotropic gels in conjunction withsodium citrate-based anticoagulant solutions, as mentioned above, do notperform well. Adequate separation and isolation of mononuclear cells formany applications cannot be achieved due to contamination with red bloodcells and granulocytes. Since red blood cells and granulocytes mustmigrate through the thixotropic gel layer during centrifugation, it hasbeen postulated that the currently available sodium citrate-basedanticoagulant solutions have somehow undesirably interacted with themigration of red blood cells, resulting in an ineffective separation ofthe red blood cell component from the mononuclear layer.

It is therefore an object of the present invention to provide a novelanticoagulant solution for use in blood chemistry-related techniquesperformed in laboratories and clinics, as well as devices employing thesame.

It is also an object of the present invention to provide a new sodiumcitrate-based anticoagulant solution for use in bloodseparation/collection containers, whereby improved separation of themononuclear cell layer is achieved upon separation by centrifugation.

It is a further object of the present invention to provide a new bloodseparation assembly including a thixotropic gel layer in conjunctionwith a sodium citrate-based anticoagulant solution, whereby an improvedseparation of the mononuclear cell layer from the remainder of a bloodsample is achieved upon centrifugation of the assembly.

SUMMARY OF THE INVENTION

The present invention is a novel anticoagulant solution for use in bloodchemistry-related techniques and devices, especially blood collectionand separation assemblies. The solution of the present invention isparticularly useful in blood separation assemblies which are capable ofcentrifugally separating lymphocytes and monocytes from heavier phasesof a sample of whole blood or, alternatively, from a pretreated cellfraction of blood. More particularly, the anticoagulant solution of thepresent invention is especially useful when employed in a bloodseparation assembly including a container, preferably a blood collectiontube, which has a thixotropic gel-like substance positioned within it.The solution is placed in the tube above the thixotropic gel. The gelcan serve as a barrier to isolate the solution from the remainder of thecomponents of the tube or as a density separation medium, or both.

The anticoagulant solution of the present invention should include aneffective concentration of sodium citrate sufficient for preventing thecoagulation of a sample of blood when such a sample is added to thecontainer. In particular, the sodium citrate-based anticoagulantsolution should have a pH ranging from above 6.0 to about 8.5 and asodium citrate concentration ranging from about 0.05M to about 0.2M.

As previously mentioned, the anticoagulant solution of the presentinvention may also be incorporated into a particular blood separationassembly, thereby providing for a new and useful version of such adevice. Such devices typically include a container having an open and aclosed end. The container is preferably a blood separation tube. Thecontainer may include a first layer of a thixotropic gel-like substancepositioned within it at a first position. The anticoagulant solution ofthe present invention should be positioned in the container at a secondposition which is closer to the open end of the container than is thefirst layer of thixotropic gel. The solution should have an effectiveconcentration of sodium citrate suitable for preventing coagulation of asample of blood when such a sample is added to the container. Inparticular, the solution should have a pH ranging from above 6.0 toabout 8.5 and a sodium citrate concentration ranging from about 0.05M toabout 0.2M. Additionally, a free space is provided adjacent to the openend of the container for receiving the blood sample and any desiredreagents.

The present invention is also directed to a new method for separatingmonocytes and lymphocytes from heavier phases of a sample of whole bloodor, alternatively, from a pretreated cell fraction of blood. A preferredembodiment of the method includes the steps of providing a containerhaving an open end and a closed end. A first layer of a thixotropicgel-like substance is positioned within the container at a firstposition. An anticoagulant solution prepared in accordance with thepresent invention and suitable for preventing coagulation of a bloodsample is introduced into the container. The solution is positioned at asecond position within the container in closer proximity to the open endof the container than the first layer. In particular, the solutionincludes an effective concentration of sodium citrate suitable forpreventing coagulation of a blood sample when such a sample is added tothe container. The solution has a pH ranging from above 6.0 to about8.5. Subsequently, a blood sample is introduced into the container andcentrifugation is performed to induce separation of lymphocytes andmonocytes from the heavier phases of the blood sample.

As previously mentioned, currently available sodium citrate-basedanticoagulant formulations have not provided satisfactory results whenused in many of the currently available blood separation/collectioncontainers. This is especially true of blood separation tubes whichutilize a thixotropic gel as a barrier or a separation means. Inparticular, the resulting layer of mononuclear cells appearing aftercentrifugation is contaminated to an unacceptable level with red bloodcells, thereby rendering the separation unacceptable for manyapplications.

Surprisingly, the sodium citrate solution of the present inventionaffords a remarkably high recovery (approximately 70-75%) of mononuclearcells when utilized as an anticoagulant in blood separation tubes havingthixotropic gel layers incorporated therein. Additionally surprising, isthe fact that red blood cell contamination is remarkably low (less than15%). These recovery and contamination levels are even more surprisingin view of the fact that the sodium citrate-based anticoagulant solutionhas a pH ranging from above 6.0 to about 8.5. This range isapproximately 1 to 2 pH points above similar sodium citrate-basedsolutions used in the past. The effectiveness of the solution of thepresent invention is indeed surprising since the highest pH at whichsodium citrate has been used in the past as an anticoagulant has been6.0.

Accordingly, the present invention provides an improved sodiumcitrate-based anticoagulant solution for use in blood chemistry-relatedtechniques and devices, especially blood collection and/or separationassemblies. For a better understanding of the present invention,together with other and further objects, reference is made to thefollowing description, the scope of which will be pointed out in theappended claims.

BRIEF DESCRIPTION OF THE FIGURE

The FIGURE illustrates a sodium citrate-based anticoagulant solutiondeployed within a blood collection and separation assembly.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the preferred embodiment of the present invention, asodium citrate-based anticoagulant solution is prepared in the followingmanner.

Trisodium citrate.2H₂ O and citric acid.H₂ O are dissolved in water inamounts sufficient to yield a sodium citrate solution having a desiredconcentration and pH which fall within the ranges set forth below. Forexample, a 0.1M sodium citrate solution having a pH of 7.0 (±0.15) canbe prepared by dissolving 29.4 grams of Na₃ citrate.2H₂ O and 0.27 gramsof citric acid.H₂ O in a liter of H₂ O.

The concentration of the sodium citrate-based solution should besufficient for preventing coagulation of a blood sample either added toa blood separation/collection device or involved in some otherlaboratory/clinical technique. In particular, the concentration ofsodium citrate should range from about 0.05M to about 0.2M, andpreferably from about 0.08M to about 0.13M. The most preferred range isfrom about 0.09M to about 0.11M.

Additionally the pH of the final sodium citrate-based solution rangesfrom above 6.0 to about 8.5, and preferably from about 6.5 to about 7.5.In the most preferred embodiment, the pH ranges from about 6.85 to about7.15.

Once the solution of the present invention has been prepared accordingto the aforementioned steps, the solution may be either employed in somelaboratory technique or added to any of several blood separation and/orcollection tubes available in the art for separating lymphocytes andmonocytes from heavier phases of whole blood or a pretreated cellfraction thereof.

While the sodium citrate-based anticoagulant solution of the presentinvention can be used for any blood separation device, it affords thegreatest advantages when used with those devices utilizing a thixotropicgel layer employed either as a cell density separation medium or as abarrier means for isolating various components of the device prior tocentrifugation.

In particular, the solution of the present invention can be employed inthe construction of an improved blood separation assembly. The preferredembodiment of the assembly includes a container having a closed end andan open end. The container is preferably of the type known in the artcapable of collecting a blood sample and undergoing subsequentcentrifugation for separation of the sample. Referring to the Figure, ablood collection and separation assembly 10 is shown. The assemblyincludes a container or tube 12 and a thixotropic gel layer 14positioned within the tube at a first position.

A variety of thixotropic gels known in the art may be used in the tubedepending upon the desired operation to be performed. For example, ifthe thixotropic gel is employed as a separation medium as well as abarrier means, the gel should have a specific gravity between 1.055 toabout 1.080 g/cm³, and preferably a specific gravity of about 1.060 toabout 1.065 g/cm³.

If the thixotropic gel is employed in conjunction with a liquid densitygradient material, the gel primarily functions as a temporary barriermeans prior to centrifugation. In such an assembly, the gel maintainsisolation of a blood sample delivered to the tube from the liquiddensity gradient material residing in the tube until analysis can beperformed at a later time. In such a situation, the specific gravity ofthe thixotropic gel should be within a sufficient range for allowingadequate separation of the mononuclear cell layer from the othercomponents of the blood sample. Preferably, the thixotropic gel employedin such an assembly has a specific gravity ranging from about 1.075 toabout 1.085 g/cc. Thixotropic gels are well-known in the art and aretypically water insoluble and chemically inert to blood. They arecommonly formulated from a dimethyl polysiloxane or polyester and aprecipitated methylated silica, wherein the methylation renders thematerial hydrophobic.

The preferred embodiment of the improved blood separation assembly ofthe present invention also includes a suitable liquid density separationmedium employed within the container at a second position which isfurther away from the open end of the container than is the thixotropicgel layer. Referring to the FIGURE, liquid density separation medium 16is shown positioned immediately below thixotropic gel layer 14.Typically, the liquid density separation medium will be of a suitabletype known in the art for separating mononuclear cells from whole blood,an example of such being Ficoll-Paque™. In addition to, or, as analternative to the liquid density separation medium mentioned above, aNewtonian gel may be employed within the tube as well.

Finally, the anticoagulant sodium citrate-based solution of the presentinvention is positioned above the thixotropic gel layer so that it mayadequately contact a whole blood sample introduced into the tube forcentrifugation and subsequent isolation of the mononuclear cell layer.Referring again to the FIGURE, anticoagulant solution 18 is shownpositioned above thixotropic gel layer 14 in closer proximity to theopen end of the separation tube 10 than the gel.

As previously mentioned, anticoagulant solution 18 should have aneffective concentration of sodium citrate sufficient for preventingcoagulation of a sample of blood when such sample is later added to thetube for centrifugation and subsequent analysis. The anticoagulantsolution should have a pH ranging from above 6.0 to about 8.5, andpreferably from about 6.5 to about 7.5. Most preferably, the pH of thesolution should range from about 6.85 to about 7.15. Optimally, the pHshould be 7.0.

In the preferred embodiment of the improved blood separation assembly ofthe present invention, the concentration of sodium citrate should rangefrom about 0.05M to about 0.2M, and preferably from about 0.08M to about0.13M. Most preferably, the concentration of sodium citrate should befrom about 0.09M to about 0.11M. Optimally, the concentration should be0.1M.

While the anticoagulant solution of the present invention is primarilycomposed of sodium citrate, additional reagents may be added, such ascell-sustaining solutions or other reagents, in order to provideadditional properties to the solution.

The preferred embodiment of the improved blood separation assembly ofthe present invention also includes a free space adjacent to the openend of the container or tube which is of a sufficient volume to receivea sample of whole blood or a fraction thereof, either alone or inconjunction with an added reagent. In particular, the FIGURE shows freespace 20 positioned above anticoagulant solution 16 in order to providesuitable space for accommodation of a blood sample to be separated.

Additionally, the assembly of the present invention may optionallyinclude a closure means for sealing the open end of the container ortube. Typically, the closure means will be suitable for providing vacuumsealing of the open end of the container as well as being pierceable bya needle in order to adapt the container for drawing a sample of bloodfrom a test subject. Referring to the FIGURE, a closure means 22 isprovided in the open end of the container or tube for creating a vacuumsealing of the container as mentioned above.

Upon the addition of a blood sample to the assembly 10, mixing of thesample with the anticoagulant solution 18 occurs, typically by manualinversion of the container 12. The thixotropic gel layer 14 remains in atemporarily fixed, first position in the tube to serve as a barriermeans for isolating the blood sample/anticoagulant solution suspensionfrom any contact with the other components of the assembly, such as theliquid density separation medium 16.

Upon the subsequent centrifugation of assembly 10, the thixotropic gellayer 14 migrates from the first position toward the top end ofcontainer 12. As centrifugation proceeds, the red blood cells separatefrom the mononuclear cell fraction and become concentrated in a layerimmediately above the thixotropic gel. As the thixotropic moves toward anew position within the tube, the red blood cells migrate through thegel to displace the liquid density separation medium below. As theliquid density separation medium is displaced, it moves upward throughthe thixotropic gel to mix with the mononuclear cellfraction/anticoagulant solution. Red blood cells and granulocytes arepelleted toward the bottom of the tube while the lymphocytes andmonocytes form a highly purified mononuclear cell layer immediatelyabove the thixotropic gel layer, thereby facilitating isolation andsubsequent removal of the mononuclear cells.

Finally, the present invention includes a method for separatinglymphocytes and monocytes from heavier phases of a sample of whole bloodor a pretreated cell fraction thereof. The method includes the steps ofproviding a container having an open and a closed end. Preferably, thecontainer is a blood collection/separation tube of the type mentionedabove. The method includes introducing a first layer of a thixotropicgel-like substance into the container or tube at a first position. Themethod further includes introducing an anticoagulant solution into thecontainer at a second position in closer proximity to the open end ofthe container than the first thixotropic gel layer. The method alsoincludes the steps of limiting the pH of the solution to any of thepreferred ranges previously mentioned as well as the step of introducingthe anticoagulant solution of the present invention into the containerwith an effective concentration of sodium citrate sufficient forpreventing coagulation of a blood sample. Additionally, the methodincludes the step of limiting the concentration of sodium citrate in thesolution to any of the ranges previously mentioned in the description ofthe anticoagulant solution.

The method of the present invention also includes the step ofintroducing a sample of whole blood or a pretreated cell fraction ofblood into the container and the subsequent step of centrifuging thecontainer to induce separation of lymphocytes and monocytes from heavierphases of the sample.

The following Example sets forth performance results for a bloodseparation assembly employing the sodium citrate solution of the presentinvention. Accordingly, the following Example serves to provide furtherappreciation of the invention but is not meant in any way to restrictits effective scope.

EXAMPLE

Forty-five blood specimens from thirty-two healthy adult volunteers wereobtained. The blood specimens were from twenty-eight male and seventeenfemale donors. Blood from each donor was collected into a LeucoPREP™brand cell separation tube with sodium citrate prepared in accordancewith the present invention (Becton Dickinson VACUTAINER Systems,Prototype Factory Work Order #2324, Lot OL178, exp. November 91, nominaldraw volume 4.0 mL) and inverted several times. For subsequentcomputation of recovery, the actual final volume of blood and citratewas measured with a pipette to the nearest 0.1 mL. Within one hour ofblood collection, the tube was centrifuged for 20 minutes at 2280 RPM(1500 xg) (SORVAL RC3C, Du Pont, Wilmington, Del.) at room temperature(25° C., ±1° C.). Following centrifugation, the LeucoPREP™ tube wasinverted eight times to resuspend the mononuclear cells in the fluidphase above the gel barrier. The fluid was quantitatively transferred toa 12×75 mm polystyrene round-bottom tube with cap (Becton DickinsonLabware, Lincoln Park, N.J., Catalog #2058). The mean percent recoveryof mononuclear cells for the LeucoPREP™ tube containing the sodiumcitrate solution from this donor population was 71.7% (±10.52). Using asingle tailed t-test, the mean percent recovery was shown to besignificantly greater than the pre-established minimum acceptablerecovery level of 50%. The mean absolute recovery of mononuclear cellsusing the LeucoPREP™ tube was 6.54×10⁶ (±1.99×10⁶) cells per tube. Therange was 3.36 to 10.54×10⁶ cells per tube. The mean volume of plasmaand liquid medium above the gel barrier was 3.2 mL (±0.22), giving amean cell concentration of 2.04×10⁶ cells per milliliter of cellsuspension. The mean recovery of mononuclear cells per mL of whole bloodwas 1.71×10⁶ from a sample with a mean mononuclear cell count of2.38×10⁶. The mean purity of the recovered white blood cell suspensionwas 98.0% (±1.79) mononuclear cells. The LeucoPREP™ results weresignificantly greater than the pre-established acceptable purity levelof 85%. The mean viability of the recovered cell suspension using theLeucoPREP™ tube was 99.9% (±0.27). The mean viability of cells recoveredusing LeucoPREP™ was significantly greater than the pre-establishedacceptable minimum of 90%. The mean percentage of red blood cellcontamination for the LeucoPREP™ was tube was 14.5% (±9.42).

While there have been described what are presently believed to be thepreferred embodiments of the invention disclosed herein, those skilledin the art will realize that changes and modification may be madethereto without departing from the spirit of the invention and it isintended to claim all such changes and modification as fall within thetrue scope of the invention.

What is claimed is:
 1. A method for separating lymphocytes and monocytesfrom heavier phases of a sample of whole blood or a pretreated cellfraction thereof, said method comprising the steps of:providing acontainer having an open end a closed end, said container further havinga first layer of a thixotropic gel-like substance contained therein at afirst position, said container also having an anticoagulant solution forpreventing coagulation of said sample when said sample is introducedinto said container, said solution located at a second position withinsaid container in closer proximity to said open end of said containerthan said first layer, said solution having an effective concentrationof sodium citrate and pH adjustor to modify the pH of the sodium citrateanticoagulant solution, sufficient for preventing coagulation of saidsample when said sample is added to said container, said solution havingan adjusted pH ranging from 6.5 to 8.5; introducing said sample intosaid container; and centrifuging said container to induce separation oflymphocytes and monocytes from heavier phases of said sample, whereinthe anticoagulant solution affords more efficient cell separation havingreduced red blood cell contamination and enhanced recovery and viabilityof the separated lymphocytes and monocytes.
 2. A method according toclaim 1, wherein said step of providing said container includes the stepof introducing said first layer of said thixotropic gel-like substanceinto said container at said first position.
 3. A method according toclaim 1, wherein said step of providing said container includes the stepof introducing said anticoagulant solution into said container at asecond position within said container in closer proximity to said openend of said container than said first layer and providing said solutionwith an effective concentration of sodium citrate sufficient forpreventing coagulation of said sample.
 4. A method according to claim 1,wherein said step of providing said container includes the step oflimiting said pH of said solution to a range from about 6.5 to about7.5.
 5. A method according to claim 4, wherein said step of providingsaid container includes the step of limiting said pH of said solution toa range from about 6.85 to about 7.15.
 6. A method according to claim 1,wherein said step of providing said container includes the step oflimiting the concentration of sodium citrate to a range from about 0.05Mto about 0.20M.
 7. A method according to claim 6, wherein said step ofproviding said container includes the step of limiting the concentrationof sodium citrate to a range from about 0.08M to about 0.13M.
 8. Amethod according to claim 1, wherein said step of providing saidcontainer includes the step of limiting the concentration of sodiumcitrate to a range from about 0.09M to about 0.11M.
 9. A methodaccording to claim 8, wherein said step of providing said containerincludes the step of limiting said pH to 7.0 and the step of limitingsaid concentration of sodium citrate to 0.1M.
 10. A method forcentrifugally separating lymphocytes and monocytes from heavier phasesof a sample of whole blood or a pre-treated cell fraction thereof, saidmethod comprising:provide a container suitable for centrifugation andhaving disposed therein a liquid density gradient material, a bloodsample and a solution having an adjusted pH ranging from 6.5 to 8.5 andcomprising an effective concentration of sodium citrate and pH adjustorto modify the pH of the sodium citrate anticoagulant solution,sufficient for preventing coagulation of said sample; and, centrifugingsaid container to induce separation of lymphocytes and monocytes fromheavier phases of said sample, wherein the anticoagulant solutionaffords more efficient cell separation having reduced red blood cellcontamination and enhanced recovery and viability of the separatedlymphocytes and monocytes.